Probing Subunits Interactions in KATP Channels Using Photo-Crosslinking via Genetically Encoded p-Azido-l-phenylalanine

Abstract

Potassium channels are multimeric protein complexes regulated by diverse physiological and pharmacological ligands. The key to understanding mechanisms of channel regulation is the ability to detect structural changes associated with ligand binding. While high-resolution structural methods such as X-ray crystallography and single-particle cryo-electron microscopy offer direct visualization of channel structures, these methods do have limitations and may not be suitable for the question of interest. In this chapter, we describe the use of a photo-cross-linker unnatural amino acid, p-azido-l-phenylalanine, to probe interactions between two proteins, the sulfonylurea receptor 1 and the inwardly rectifying potassium channel Kir6.2, that form the ATP-sensitive potassium (KATP) channel complex in the absence or presence of ligands. The difference in the extent of crosslinking between a liganded state and unliganded state can be used as a readout of ligand-induced structural changes. We anticipate that the protocol described here will also be applicable for other potassium channels and protein complexes.